Site-specific recombination in eukaryotes and constructs useful therefor

ABSTRACT

Site-specific recombinases provide a means of efficiently manipulating chromosomal sequences in mammalian cells in culture and in mice. Embryonic stem cells containing recombinase nucleic acid constructs that were expressed in the male germline would simplify current protocols for producing mice bearing homologously recombined alleles that have been secondarily rearranged by a site-specific recombinase, five lines of transgenic mice containing a fusion gene consisting of the mouse protamine  1  gene promoter and the Cre recombinase coding sequence (ProCre nucleic acid constructs) showed high levels of Cre-mediated recombination of the germline, but did not show appreciable recombination in other tissues. In different ProCre strains, between 80% and 100% of the progeny that inherited a Cre target nucleic acid construct from males that were also heterozygous for a ProCre nucleic acid construct inherited the Cre-recombined target. ProCre nucleic acid constructs and recombined targets segregated in the first generation. When ES cells prepared from one ProCre line were transfected with vectors containing a loxP-flanked neomycin cassette, G418 resistant, homologously recombined clones, in which the loxP sites remained functional, were readily isolated. These data establish that ProCre nucleic acid constructs will facilitate the production of subtle, conditional, or tissue-specific mutations in mice as well as the production and analysis of mice with recombinase-conditional lethal alleles.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/437,556, filed May 19, 2006 which is a division of U.S. patentapplication Ser. No. 08/919,501, filed Aug. 28, 1997, now U.S. Pat. No.7,135,608, the entire contents of each of which are hereby incorporatedby reference herein for all purposes.

FIELD OF THE INVENTION

The present invention relates to methods for manipulating chromosomalsequences in cells by site-specific recombination promoted byrecombinases. In a particular aspect, the present invention relates tomethods for producing embryonic stem cells bearing nucleic acidsequences that have been rearranged by a site-specific recombinaseexpressed from a construct controlled by a tissue-specific promoter(e.g., a germline specific promoter). In another aspect, the presentinvention relates to methods for producing embryonic stem cells bearingnucleic acid sequences that have been rearranged by a site-specificrecombinase expressed from a construct controlled by a conditionalpromoter.

BACKGROUND OF THE INVENTION

The analysis of gene function has increasingly come to require theproduction of subtle, tissue-specific, and conditional mutations inanimals and plants. Although there are a number of methods forengineering subtle mutations in embryonic stem (ES) cells (Hasty et al.(1991) Nature 350:243-246, Askew et al. (1993) Mol Cell Bio)13:4115-4124), the use of site-specific recombinases to remove theselectable marker that permits isolation of homologously recombined EScell clones has become increasingly prevalent (Kitamoto et al. (1996)Biochem Diophys Res Commun 222:742-747, Fiering et al. (1993) Proc NatlAcad Sci USA 90:8469-8473, Schwenk et al. (1995) Nucleic Acids Res23:5080-5081; Gu et al. (1993) Cell 13:1155-1164; Sailer et al. (1996)Taniguchi Symposia on Brain Sciences, eds. Hakanishi et al. (JapanScientific Press), pp. 89-98).

Site-specific recombinases represent the best method for creatingtissue-specific and conditional mutations in animals and plants beingemployed first to remove the selectable marker to create a functionallywild-type allele, and then to inactivate the allele mosaically inanimals and plants by removing some essential component in atissue-specific or conditional manner (Ciu et al. (1994) Science265:103-106; Kuhn et al. (1995) Science 269:1427-1429). Currentprotocols for using excessive site-specific recombination to removeselectable markers include transiently transfecting ES cell clones witha recombinase expression vector (Gu et al., (1993) Cell 73:1155-1164),microinjecting fertilized oocytes containing the recombinant allele witha recombinase expression vector (Kitamoto et al. (1996) Biochem BiophysRes Commun 222:742-747; Araki et al. (1995) Proc Natl Acad Sci USA92:160-164), or breeding animals and plants containing the recombinantallele to animals and plants, respectively, containing a recombinasetransgene (Schwenk et al. (1995) Nucleic Acids Res 23:5080-5081;Lewandoski et al. (1997) Curr Biol 7:148-151). Each of these approachesrequires an investment of some combination of time, resources, andexpertise over that required to generate animals and plants withhomologously recombined alleles. The most commonly employed method, thesecondary transfection of homologously recombined ES cell clones with arecombinase expression vector, additionally requires extended culturetime that may decrease their potential to enter the germline.

In principle, marker excision would be substantially simplified throughthe use of ES cells containing recombinase nucleic acid constructs thatwere expressed in the germline, but not to an appreciable extent in theES cells themselves or somatic tissues of animals and plants. The lackof ES cell expression would mean that targeting vectors containingselectable markers flanked by recombinase target sites could be used toisolate homologous recombinants without fear that the marker would beexcised during culture. Robust recombinase expression in gametes wouldmean that the marker would be excised in at least some of the progeny ofES cell chimeras. Only a single step would be required to isolate subtlemutations and, if two different recombinase systems were employed,conditional and tissue-specific alleles could be produced with similarimprovements in efficiency. A germline-specific recombinase nucleic acidconstruct could also be used to deliver recombined target nucleic acidconstructs to the early embryo (Lewandoski et al. (11997) Curr Biol7:148-151), so long as the recombined target was not detrimental to theterminal stages of spermatogenesis.

Previous reports have shown that expression of nucleic acid constructscontaining the proximal promoter of the mouse protamine 1 (mP1) locus isrestricted to haploid spermatids in mature mice (Peschon et al. (1987)Proc Natl Acad Sci USA 84:5316-5319; Behringer et al. (1988) Proc NatlAcad Sci USA 85: 2648-2652) although low levels of ectopic expressionmay occur in some mature tissues (Behringer et al. (1988) Proc Natl AcadSci USA 85:2648-2652). Inclusion of the mP1 promoter does not guaranteeexpression in the male germline, however, for although nucleic acidconstructs containing the mP1 promoter and the SV40 T-antigen codingsequence were transcribed, the message was not translated at detectablelevels in spermatids (Behringer et al. (1988) Proc Natl Acad Sci USA 85:2648-2652).

Accordingly, there is a need in the art for methods to modulateexpression of recombined target nucleic acid sequences in the earlyembryo. In addition, there is a need in the art for tissue-specific andconditional recombinatory tools to create transgenic animals and plants.These and other needs in the art are addressed by the present invention.

BRIEF DESCRIPTION OF THE INVENTION

The present invention meets the need in the art for modulatingexpression of recombined target nucleic acid sequences to the earlyembryo. The present invention further meets the need in the art fortissue-specific and conditional recombinatory tools to create transgenicanimals and plants. Thus, in accordance with the present invention, ithas been discovered that nucleic acid constructs encoding a germlinespecific promoter operatively associated with a recombinase codingsequence lead to efficient recombination of a target nucleic acidconstruct in the male germline, but not in other tissues. This suggeststhat such nucleic acid constructs could be used for the efficientproduction of embryos bearing conditional, genetically lethal alleles.It has additionally been discovered that ES cell lines generated fromone of these transgenic lines could be used in combination withtargeting vectors that contained loxP-flanked selectable markers toisolate homologous recombinants containing the marker and functionalloxP sites.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a schematic of P2Bc and P2Br alleles. The positionsof the PCR primers used (5′P and 3′P) are indicated on the diagrams ofthe P2Bc and P2Br alleles.

FIG. 2 depicts the targeting of the hoxb-1 locus in ProCre ES cellsusing a targeting vector that contains a loxP-flanked selectable marker.Top, schematic of the wild-type hoxh-1 locus showing the positions ofthe two exons (open boxes), the position of a 5′ NruI site and flankingBamHI restriction endonuclease sites, and PCR primers (triangles) thatamplify a 204 bp product from the wild-type allele that includes theNruI site. Middle, the predicted organization of homologously recombinedhoxb-1 allele in which a neomycin cassette (NEO), flanked by loxP sites(L), bias been inserted into the NruI site shown in the top diagram. Theinsertion creates a novel BamHI site and the same PCR primers nowamplify a 1600 bp product. Bottom: the predicted structure of therecombined allele shown in the middle panel after Cre-mediated excisionof the neomycin cassette to leave a single loxP site in place of theNruI site of the wild-type allele. Amplification with the same primersnow yields a 268 hp product.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided nucleicacid constructs comprising a germline-specific promoter operativelyassociated with a recombinase coding sequence.

As used herein, the term “promoter” refers to a specific nucleotidesequence recognized by RNA polymerase, the enzyme that initiates RNAsynthesis. The promoter sequence is the site at which transcription canbe specifically initiated under proper conditions. The recombinasenucleic acid(s), operatively linked to the suitable promoter, is (are)introduced into the cells of a suitable host, wherein expression of therecombinase nucleic acid(s) is (are) controlled by the promoter.

Germline-specific promoters contemplated for use in the practice of thepresent invention include the protamine 1 gene promoter, the protamine 2gene promoter, the spermatid-specific promoter from the c-kit gene(Albanesi et al. (1996) Development 122(4): 1291-1302), the spermspecific promoter from angiotensin-converting enzyme (Howard et al.(1993) Moll Cell Biol 13(1):18-27; Zhou et al. (1995) Dev Genet.16(2):201-209), oocyte specific promoter from the ZP2 gene, oocytespecific promoter from the ZP2 gene, oocyte specific promoter from theZP3 gene (Sehickler et al. (1992) Moll Cell Biol 12(1):120-127), and thelike.

In addition to the above-described germline-specific promoters,tissue-specific promoters specific to plants are also contemplated foruse in the practice of the present invention, including, for example,the LAT52 gene promoter from tomato, the LAT56 gene promoter fromtomato, the LAT59 gene promoter from tomato Eyal et al. (1995) PlantCell 7(3):373-384), the pollen-specific promoter of the Brassica S locusglycoprotein gene (Dzelzkalns et al. (1993) Plant Cell 5(8):855-863),the pollen-specific promoter of the NTP303 gene (Weterings et al. (1995)Plant J 8(1):55-63), and the like.

Recombinases contemplated for use in the practice of the presentinvention include Cre recombinase, FLP recombinase, the R gene productof Zygosaccharomyces (Onouchi et al. (1995) Mol Gen Genet. 247(6):653-660), and the like.

Presently preferred constructs contemplated for use in the practice ofthe present invention include ProCre (comprising the protamine 1 genepromoter operatively associated with Cre recombinase), ProFLP(comprising the protamine 1 gene promoter operatively associated withFLP recombinase). ProR (comprising the protamine 1 gene promoteroperatively associated with the R gene product of Zygosaccharomyces),and the like.

In accordance with another embodiment of the present invention, thereare provided nucleic acid constructs comprising a conditional promoteror a tissue-specific promoter operatively associated with a recombinasecoding sequence.

Promoters contemplated for control of expression of recombinase nucleicacid(s) employed in accordance with this aspect of the present inventioninclude inducible (e.g., minimal CMV promoter, minimal TK promoter,modified MMLV LTR), constitutive (e.g., chicken β-actin promoter, MMLVLTR (non-modified), DHFR), and/or tissue specific promoters.

Conditional promoters contemplated for use in the practice of thepresent invention comprise transcription regulatory regions thatfunction maximally to promote transcription of mRNA under inducingconditions. Examples of suitable inducible promoters include DNAsequences corresponding to: the E. coli lac operator responsive to IPTC(see Nakamura et al., Cell, 18:1109-1117, 1979), the metallothioneinpromoter metal-regulatory-elements responsive to heavy-metal (e.g.,zinc) induction (see Evans et al., U.S. Pat. No. 4,870,009), the phageT71ac promoter responsive to IPTG (see Studier et al., Meth Enzymol.,185:60-89, 1990; and U.S. Pat. No. 4,952,496), the heat-shock promoter;the TK minimal promoter; the CMV minimal promoter; a synthetic promoter;and the like.

Exemplary constitutive promoters contemplated for use in the practice ofthe present invention include the CMV promoter, the SV40 promoter, theDHFR promoter, the mouse mammary tumor virus (MMLV) steroid-induciblepromoter, Moloney murine leukemia virus (MMLV) promoter, elongationfactor Iα (EFIα) promoter, albumin promoter, APO A1 promoter, cyclic AMPdependent kinase II (CaMKII) promoter, keratin promoter, CD3 promoter,immunoglobulin light or heavy chain promoters, neurofilament promoter,neuron specific enolase promoter, L7 promoter, CD2 promoter, myosinlight chain kinase promoter, HOX gene promoter, thymidine kinase (TK)promoter, RNA Pol II promoter, MYOD promoter, MYF5 promoter,phophoglycerokinase (PGK) promoter, Stfl promoter, Low DensityLipoprotein (LDL) promoter, chicken β-actin promoter (used inconjunction with ecdysone response element) and the like.

As readily understood by those of skill in the art, the term “tissuespecific” refers to the substantially exclusive initiation oftranscription in the tissue from which a particular promoter, whichdrives expression of a given gene, is derived (e.g., expressed only inT-cells, endothielial cells, smooth muscle cells, and the like).Exemplary tissue specific promoters contemplated for use in the practiceof the present invention include the GH promoter, the NSE promoter, theGFAP promoter, neurotransmitter promoters (e.g., tyrosine hydroxylase,TH, choline acetyltransferase, ChAT, and the like), promoters forneurotropic factors (e.g., a nerve growth factor promoter, NT-3, BDNFpromoters, and the like), and so on.

In accordance with yet another embodiment of the present invention,there are provided embryonic stem cells containing a nucleic acidconstruct as described herein.

As readily understood by those of skill in the art, the above-describedconstructs can be introduced into a variety of animal species. such as,for example, mouse, rat, rabbits, swine, ruminants (sheep, goats andcattle), humans, poultry, fish, and the like. Transgenic amphibians,insects, nematodes, and the like, are also contemplated. Members of theplant kingdom, such as, for example, transgenic mono- and dicotyledonousspecies, including important crop plants, i.e., wheat, rice, maize,soybean, potato, cotton, alfalfa and the like, are also contemplated.

For example, pluripotential ES cells can be derived from earlypreimplantation embryos, preferably the ova are harvested between theeight-cell and blastocyst stages. ES cells are maintained in culturelong enough to permit integration of the promoter-recombinase nucleicacid construct(s). The cells are then either injected into a hostblastocyst, i.e, the blastocoel of the host blastocyst, or coculturedwith eight-cell to morula-stage ova, i.e, zona-free morula, so thattransfected ES cells are preferentially incorporated into the inner cellmass of the developing embryo. With blastocyst injection, transgenicoffspring are termed “chimeric,” as some of their cells are derived fromthe host blastocyst and some tracisfected ES cells. The host embryos aretransferred into intermediate hosts or surrogate females for continuousdevelopment.

The transformation procedure for plants usually relies on the transferof a transgene carrying a particular promoter construct via the soilbacterium Agrobacterium tumefaciens. Transformation vectors for thisprocedure are derived from the T-DNA of A. tumefaciens, and transgenesare stably incorporated into the nuclear genome. The activity of thetransgenes can then be monitored in the regenerated plants underdifferent conditions. In this way, many promoter elements that areinvolved in complex regulatory pathways such as light responsiveness ortissue specificity have been defined.

Alternatively, direct (i.e., vectorless) gene transfer systems are alsocontemplated including chemical methods, electroporation,microinjection, biolistics, and the like. Protoplasts isolated from theplants can be obtained by treatment with cell wall degrading enzymes-DNAcan be introduced into plant protoplasts by a number of physicaltechniques including electroporation and polyethylene glycol treatmentin the presence of MgCl. The method of choice for rapid promoteranalyses in plants is the biolistic method. This technique involves thedelivery of the particular DNA construct into plant cells bymicroprojectiles, i.e., nucleic acid(s) coated or precipitated bytungsten or gold. This method is not limited to any particular plantspecies or tissue type. Preferably, this method would allow quantitativeanalysis of transformation if appropriate selectable markers areincluded.

In a preferred embodiment, the genome of embryonic stern cells accordingto the invention comprise a transcriptionally active selectable markerflanked by two recombination target sites. It is especially preferredthat the recombinase encoded by the recombinase coding sequenceoperatively associated with a germline-specific promoter is selectivefor the recombination target sites flanking said selectable marker.

Optionally, embryonic stem cells according to the invention may furthercomprise one or more of:

a nucleic acid fragment flanked by two recombination target sites,wherein said recombination target sites are different than therecombination target sites which flank said selectable marker.

a nucleic acid construct comprising a conditional promoter operativelyassociated with a recombinase coding sequence.

a second nucleic acid construct comprising a tissue-specific promoteroperatively associated with a second recombinase coding sequence, or thelike. Preferably, the second recombinase coding sequence will bedifferent than the first recombinase coding sequence.

The ability to select and maintain nucleic acid constructs in the hostcell is an important aspect of an expression system. The most importanttype of selectable marker incorporated in the nucleic acid construct isan antibiotic resistance element allowing selection with ampicillin,kanamycin, neomycin, tetracycline, hygromycin, puroncycin, blastophycin,and the like. Other approaches employ specially constructed host cellswhich require the selectable marker for survival. Such selectablemarkers include the valine tRNA synthetase, vat S, the single-strandedDNA binding protein, ssb, thymidine kinase, or the like. Alternatively,naturally occurring partition systems that maintain copy number andselect against plasmid loss is also contemplated. An example is theincorporation of the parB locus. Other selectable markers include HPRTand the like.

Selectable markers specific for plants include, the gus A (iud A), thebar gene, phosphinothricin and the like.

In accordance with still another embodiment of the present invention,there are provided methods for excision of the transcriptionally activeselectable marker from the above-described embryonic stem cells, saidmethod comprising:

passaging the genome derived from said embryonic stem cells throughgametogenesis (i.e., spermatogenesis or oogenesis).

Excision of marker as contemplated herein can cause a variety of endresults, e.g., deletion of the marker or a nucleic acid sequence, gainof function or loss of function, replacement of function, and the like,as well as modulation of any one or more of these results.

Functions which are contemplated to be manipulated include regulatingbody size and growth rate, including recombining gene constructs whichcontain various growth hormone gene sequences. Other productivity traitsthat are targets include altering the properties or proportions ofcaseins, lactose, or butterfat in milk, increased resistance to viraland bacterial diseases (i.e., “constitutive immunity” or germ-linetransmission of specific, recombined antibody genes), more efficientwool production, and the like. Other functions which are contemplated tobe modulated include development of lines of transgenic animals andplants for use in directing expression of transgenes encodingbiologically active human proteins.

Agronomic traits which are contemplated to be modulated by use of thepresent invention include tolerance to biotic and antibiotic stresses,increased resistance to herbicides, pest damage, and viral, bacterial,and fungal diseases, improvement of crop quality (i.e., increase innutritional value of food and feed), reduction of post-harvest losses,improvement of suitability acid enlargement of the spectrum forprocessing (i.e., altered quantity and composition of endogenousproperties, production of new compounds of plant or non-plant originsuch as biopolymers or pharmaceutical substances).

In accordance with a still further embodiment of the present invention,there are provided methods for the production of recombinant alleles,said method comprising:

introducing a nucleic acid fragment flanked by at least tworecombination target sites into embryonic stem cells as describedherein, and

passaging the genome derived from said embryonic stem cells throughgametogenesis.

As readily recognized by those of skill in the art, nucleic acidfragments can be introduced into ES cells by a variety of techniques,e.g., by homologous recombination, random insertion, retroviralinsertion, site specific-mediated recombination, and the like.

Nucleic acid fragments contemplated for use herein include fragmentscontaining an essential portion of a gene of interest.

In accordance with yet another embodiment of the present invention,there are provided methods for the production of recombinant alleles,said method comprising:

introducing at least one recombinase responsive construct into embryonicstem cells as described herein,

wherein said construct(s) comprise(s) a nucleic acid fragment and aselectable marker.

wherein said selectable marker is flanked by a first pair ofrecombination target sites, and

wherein said nucleic acid fragment is flanked by a second pair ofrecombination target sites,

passaging the genomic derived from said embryonic stem cells throughgametogenesis.

In a presently preferred aspect, this first pair of recombination targetsites is recognized by a recombinase which is expressed under thecontrol of a germline-specific promoter and said second pair ofrecombination target sites is recognized by a recombinase which isexpressed under the control of a conditional promoter or a tissuespecific promoter.

Optionally, the embryonic stem cells employed herein can furthercomprise a second nucleic acid construct selected from constructscomprising a conditional promoter operatively associated with arecombinase coding sequence, a construct comprising a tissue-specificpromoter operatively associated with a recombinase coding sequence, acidthe like.

In accordance with still another embodiment of the present invention,there are provided methods for the conditional assembly of functionalgene(s) for expression in eukaryotic cells by recombination ofindividual inactive gene segments from one or more gene(s) of interest,

wherein each of said segments contains at least one recombination targetsite, and

wherein at least once of said segments contains at least tworecombination target sites, said method comprising:

-   -   introducing said individual inactive gene segments into an        embryonic stem cell as described herein, thereby providing a DNA        which encodes a functional gene of interest, the expression        product of which is biologically active, upon passage of the        genomic derived from said stem cells through gametogenesis.        For assembly of functional genes from inactive gene segments,        see, for example, U.S. Pat. No. 5,654,182, incorporated herein        by reference in its entirety.

In accordance with a still further embodiment of the present invention,there are provided methods for the generation of recombinant livestock,said method comprising:

combining embryonic stem cells that include nucleic acid constructaccording to the invention with host pluripotential ES cells derivedfrom early preimplantation embryos, and

introducing these combined embryos into a host female and allowing thederived embryos to come to term.

In accordance with yet another embodiment of the present invention,there are provided methods for the generation of recombinant plants,said method comprising transforming plant zygotes with nucleic acidconstructs according to the invention and allowing the zygote todevelop.

The objective of the current work with ProCre nucleic acid constructswas to determine the potential of germline-specific promoters toimplement efficient approaches utilizing site-specific recombinases togenerate an array of sophisticated mutations in mammals and plants. Thedata shows that it is possible to create recombinase nucleic acidconstructs that are expressed at high levels in the germ line but not toa functionally significant extent in either ES cells or embryonic oradult somatic tissues. Homologous recombinants with a selectable markercan be isolated in ES cells that contain promoter-recombinase nucleicacid constructs. Transgenic animals and plants bearing thepromoter-recombinase nucleic acid constructs and a target alleletransmit the recombined target to their progeny at high frequencies.These results establish the principle that mammals and plants containingloci that have been homologously recombined and then subsequentlysite-specifically recombined can be generated simply by using ES cellswith a suitable recombinase nucleic acid constructs for the initialtargeting. By this mechanism, alleles containing a single recombinasetarget site and a mutation of interest can be produced in the progeny ofES cell chimeras without any investment of time, expertise, or resourcesover that required to create an allele that still contains a selectablemarker. The paradigm has obvious utility in the production of subtle andconditional mutations that require generation of alleles with minimalstructural alterations. Because the presence and transcriptionalactivity of selectable markers can contribute to phenotypes in anunanticipated and unwanted manner (Fiering et al. (1995) Genes Dev9:2203-2213); Olson et al (1996) Cell 85:14), the approach will also beuseful for generating null alleles.

Expression of the endogenous mP1 locus (Hecht et al. (1986) Exp Cell Res164:183-190), and mP1-driven nucleic acid constructs (Behringer et al.(1988) Proc Natl Acad Sci USA 85:2648-2652; Braun et al. (1989) Nature337:373-376; Zambrowicz et al. (1993) Proc Natl Acad Sci USA90:5071-5075) is restricted to haploid spermatids. Expression of mP1nucleic acid construct expression typically begins at haploid stages,and both RNA (Caldwell and Handel (1991) Proc Natl Acad Sci USA88:2407-2411) and proteins (Braun et al. (1989) Nature 337:373-376)diffuse through the spermatogenic syncytium. The result is a highlyefficient recombination of target alleles and the segregation ofrecombinase and target nucleic acid constructs in the first generation.

Cre-mediated recombination proved to be highly testis-specific in ProCremice. It is clear that the nucleic acid constructs are not expressed inthe inner cell mass or in other early embryonic tissues. Cells frompre-implantation embryos intermingle extensively and the embryo as awhole is derived from a small number of cells (Beddington et al. (1989)Development 106: 37-46; Soriano and Jaenisch (1986) Cell 46:19-29). IfProCre nucleic acid constructs recombined target sequences duringpreimplantation stages, at least a few percent of the cells in manytissues would contain the P2Br allele and Southern and PCR analysesshowed that this was not the case. The ectopic Cre activity seen in someProCrc strains probably resulted from low levels of recombinaseexpression in later embryos or mature tissues, a finding consistent withthe expression patterns of other mP1-driven nucleic acid constructs.Northern analyses have failed to reveal the expression of mP1-containingnucleic acid constructs in a variety of mature tissues (Peschon et al.(1987) Proc Natl Acad Sci USA 84:5316-5319; Behringer et al. (1988) ProcNatl Acad Sci USA 85:2648-2652; Peschon et al. (1989) Ann N Y Acad Sci564:186-197; Zambrowicz et al. (1993) Proc Natl Acad Sci USA90:5071-5075), but nucleic acid constructs containing the mP1 promoterand the SV40 T-antigen led to the consistent development of tumors ofthe petrosal bone and right cardiac atrium (Behringer et al. (1988) ProcNatl Aced Sci USA 85:2648-2652).

PCR assays represent a very sensitive assay for whether sufficientlevels of Cre protein were produced to effect recombination.Importantly, they measured the cumulative level of recombination, forevents that occurred at any stage of development are likely to have beenpropagated to, and might be amplified in, descendant populations. Thehighest level of ectopic recombination was that observed in cardiacventricular tissue of strain which generated a signal approximatelyequivalent to that expected if the ratio between recombined andunrecombined alleles were 1:104. The activities observed in otherstrains were considerably lower than this, and one strain did not showany ectopic activity. None of the strains showed evidence ofrecombination in the cardiac atria and the petrosal bone was notexamined. These assays did not rule out the possibility that higherlevels of recombination occur in tissues that were not examined or thatthe low levels of recombination observed in some tissues reflected highlevels of recombination in some component cell population.

These low levels of ectopic activity suggest that mP1-driven recombinasenucleic acid constructs could be used for the production of embryoscontaining genetically lethal alleles. Some alleles created byhomologous recombination in ES cells will prove to be lethal inheterozygotes, as was the case for mRNA editing mutation of the GluR2glutamate receptor subunit (Brusa et al. (1995) Science 270:1677-160).Germline transmission would be restricted to rare chimeras in which timelevel of chimerism was low enough in tissues affected by the mutation toallow survival and high enough in the germline to allow transmission.This problem could be circumvented by creating recombinase-conditionalmutations in ES cells bearing mp1-recombinase nucleic acid constructs,or by making the same mutations in standard ES cells and thenintroducing the mp1-recombinase nucleic acid construct by breeding. Solong as the recombined version of the allele did not adversely impactterminal stages of spermatogenesis, embryos containing the recombinedallele could be efficiently produced. Embryos containing recombinednucleic acid constructs can also be produced through the activity of Crenucleic acid constructs that are expressed during early embryogenesisfrom the human cytomegalovirus minimal promoter (Schwenk et al. (1995)Nucleic Acids Res 23:5080-5081), the adenovirus Ella promoter (Lakso etal. (1992) Proc Natl Acad Sci LSA 89:6232-6236), or the zP3 promoter(Lewandoski et al. (1997) Curr Biol 7:148-151) ProCre and zP3 nucleicacid constructs have the advantage of delivering a recombined allele tothe zygote, guaranteeing that all cells in the derived embryos willcontain the allele.

ProCre cells are but one of many different kinds of recombinase-bearingES cells that could significantly shorten the time and effort requiredfor a wide variety of genetic manipulations in mice. The most obvious ofthese are complimentary ProFLP ES cells in which the FLP recombinase wasderived from S. cerevisae (Broach and Hicks (1980) Cell 21:501-508) oranother species (Kuhn et al. (1995) Science 269:1427-1429). Conceptuallydistinct from these but perhaps as generically useful would be ES cellsbearing inducible recombinase nucleic acid constructs that wouldfacilitate temporal control of recombinase expression in ES cells,chimeras, and heir progeny to generate site-specifically recombinedalleles (Araki et al (1992) J Mol Biol 225:25-37: No et al. (1996) ProcNatl Acad Sci USA 93:3346-3351, Logie and Stewart (1995) Proc Natl AcadSci USA 92:5940-5944; Feil et al. (1996) Proc Natl Acad Sci USA93:10887-10890). Finally, fusion genes that led to recombinaseexpression in specific tissues could be used to address specificresearch objectives.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

Example 1 Mammalian DNA Constructs

A 652 bp fragment of the mP1 promoter (SEQ ID NO:1; Peschon et al.(1989) Annals of the New York Academy of Sciences 186-197) was isolatedby PCR using PCR primers (SEQ ID NOs:2 and 3) genomic DNA from CCE EScells (Robertson et al. (1986) Nature 323:445-448) as a template. Thisfragment was fused to a Cre coding sequence (SEQ ID NO:4) modified tocontain a consensus translation start site (Kozak (1986) Cell44:283-292), 11 codons for a human c-myc epitope (Evan et al. (1985) MolCell Biol 5:3610-3616), 7 codons for a minimal SV40 nuclear localizationsignal (Kalderon et al. (1984) Cell 39:499-509) and the polyadenylationsisal from pIC-Cre in the plasmid pOG304M (SEQ ID NO:5). The Creexpression plasmid pOG231 was prepared by fusing a Cre coding sequencemodified from pIC-Cre (Gu et al. (1993) Cell 73:1155-1164), andcontaining the same translation start and nuclear localization signal,to the synthetic intron and CMV promoter of pOG44 (O'Gorman et al.(1991) Science 251:1351-1355).

A plasmid, pOG277 (SEQ ID NO:7), containing a loxP-flanked neomycincassette was prepared by inserting a wild-type loxP site (SEQ ID NO:8;Hoess et al. (1982) Proc Natl Acad Sci USA 79:3398-402) into pBSKS(Stratagene) and then cloning the neomycin expression cassette frompMClneo-polyA (Thomas et al. (1987) Cell 51:503-512) between interationsof this loxP site. The hoxb-1 targeting construct consisted of thePCGK-TK cassette from pPNT (Tybulewicz et al. (1991) Cell 65:1153-63),and 1.4 kb) and 10.2 kb of sequences 5′ and 3′ to an Nru I site 800 bp5′ to the hoxb-1 transcriptional start site isolated from a 129 straingenomic library (Stratagene). The loxP-flanked neo cassette from pOG277was inserted into the NruI site. The pOG277 neomycin cassette and aβ-GAL sequence was inserted into the first exon of the large subunit ofRNA polymerase II (RP2) (Ahearn et al. (1987) J. Biol. Chem.262:10695-10705) to create the P2Bc allele (FIG. 1). Cre-mediatedrecombination of the P2Bc allele results in the deletion of the neomycincassette (Neo) of P2Bc that is flanked by two loxP sites, leaving asingle loxP sire and fusing the B-Gal coding sequence to the initialcodons of the RNA polymerase II coding sequence. Recombination increasesthe size of a Pst I fragment recognized by the RP2 probe, which isexternal to the targeting vector used, indicated by the shaded box beloweach allele.

Example 2 Production of Transgenic Mice

Fertilized oocytes obtained from matings of 129/SvJae (Simpson et al.(1997) Nat Genet. 16:19-27) and BALB/c×C57BL/6 F1 mice were used forpronuclear injections of (the Protamine-Cre fusion gene from pOG304Maccording to standard protocols (Hogan et al. Manipulating the MouseEmbryo: The Manual, Coldspring Harbor Press (1994), pg. 497). Productionof ES cells and homologous recombinants: Heterozygous ProCrc 129/Svjacmales were mated to 129/SvEms-+−^(?)/J females (Simpson et al. (1997)Nat Genet. 16:19-27) to produce blastocysts that were cultured accordingto standard protocols (Robertson (1987) Teratocarcinomas and embryonicstem cells, a practical approach, eds. E. J. Robertson (IRL Press), pp.71-112). The sex (King et al. (1994) Genomics 24:159-68) and ProCrestatus of each line were determined by PCR assays. Molecular analyses:Tail biopsy genomic DNA was used for hybridization assays or PCR assaysto identify ProCre and P2Bc/r mice. PCR reactions for the detection ofectopic Crc activity used 100 ng of genomic DNA as a template to amplifya P2Br-specific product using a 5′ primer from the RP2 promoter and a 3′primer from the β-GAL coding sequence (FIG. 1). Thirty cycles ofamplification were done in a total volume of 100 μl using 300 ng of eachprimer, 3 mM MgCl2, 1.5 units of Taq polymerase, and an annealingtemperature of 60° C. Southern blots of reaction products werehybridized with a probe specific for the P2Br reaction product.

Example 3 ProCre Nuclei Acid Constructs Efficiently Recombine TargetAlleles

A total of nine founder animals with ProCre nucleic acid constructs wereobtained from injections of a Protamine-Cre fusion gene. Two lines werederived from injections of 129SvJae (Simpson et. al. (1997) Nat Genet.16:19-27) embryos, and seven from injections of CB6F2 embryos. The129/SvJae lines and three randomly selected hybrid lines were examinedin detail. To determine whether ProCre nucleic acid constructs wouldefficiently recombine a target allele, males were generated thatcontained a ProCre nucleic acid construct and a target for Cre-mediatedrecombination. This “P2Bc” (Pol II, β-Gal, conditional) target (FIG. 1)was created using homologous recombination in ES cells to insert aloxP-flanked neomycin cassette and a β-GAL coding sequence into thefirst exon of the locus coding for the large subunit of RNA polymeraseII. Crc-mediated recombination of the loxP sites was expected to deletethe intercalated sequences, creating “P2Br” allele (Pol II, β-Gal,recombined).

These males were mated to wild-type females and the resulting progenywere examined by Southern blotting to determine if they inherited theP2Bc or the P2Br allele, and to additionally determine the segregationpattern of ProCre nucleic acid constructs anti P2Br alleles. Southernblot of Pst I digested tail biopsy DNA's from a +/P2Bc, +/ProCre male(sire) and four of his progeny by a wild-type female probed with n RP2probe (top) and then reprobed with a Cre probe (bottom). The largemajority of transmitted target alleles were Cre-recombined P2Br alleles(Table 1). ProCre nucleic acid constructs and recombined target allelessegregated independently in the first generation approximately 50% ofmice that inherited a P2Br allele also inherited their male parent'sProCre nucleic acid construct. All RP2 mutant alleles in the progenywere P2Br, and some progeny inherit a P2Br allele without inheritingProCre nucleic acid construct and is homozygous wild-type at the RP2locus. These data establish that ProCre nucleic acid constructsefficiently recombine the P2Bc allele in the male germline and that therecombined P2Br alleles and ProCre nucleic acid constructs segregate inthe first generation. Because significantly more than 25% of the progenyinherited recombined target alleles, recombination either occurredduring diploid stages of spermatogenesis or Crc generated during haploidstages of spermatogenesis was distributed among spermatids throughcytoplasmic bridges (Braun et al. (1989) Nature 337:373-376), effectingrecombination in spermatids that did not themselves contain a ProCrenucleic acid construct.

The progeny of matings between ProCre males and +/P2Bc females were alsoexamined to determine if male gametes from ProCre mice delivered throughCre to zygotes to effect Cre-mediated recombination of a targetsequence. Of 96 progeny examined by Southern blotting, none contained aCre-recombined P2Br allele.

It has also been discovered that a loxP-flanked neo cassette in theglutamate receptor P6 subunit locus is efficiently recombined by ProCrenucleic acid constructs in mice.

Example 4 ProCre Nucleic acid construct Expression is HighlyTissue-Specific

Genomic DMAs from ten different tissues of five-to seven-week old malesthat contained both a ProCre nucleic acid construct and a P2Bc targetallele were analyzed in Southern blots. Southern blots were prepared ofPst I digested DNA from testes (T) and one other tissue (K, kidney B,brain; S, spleen) of males heterozygous for one of four ProCre nucleicacid constructs and the P2Br allele. Testis DNA from each male shows aP2Br allele signal, in addition to those generated by the wild-type RP2(WT) and P2Bc alleles. Other tissues show only the WT and P28c signals.Only the testis samples showed signal indicating Cre-mediatedrecombination of the target. The intensity of the P2Br signal relativeto that of the wild-type allele ranged from 0% to 22% for differentProCre strains and did not correlate with the ProCre nucleic acidconstruct copy number. The copy number of ProCre nucleic acid constructsvaried among lines showing similar levels of recombination in testis.For example, restriction patterns and densitometric analyses showed thatline 58 contained a single copy of the ProCre nucleic acid construct,yet showed virtually the same testis recombination signal as linecontaining more than 10 copies. This variability is similar to resultsobtained with other mP1 promoter-driven nucleic acid constructs (Peschonet al. (1987) Proc Natl Acad Sci USA 84:5316-5319; Zambrowicz et al.(1993) Proc Natl Acad Sci USA 90:5071 5075).

As a more sensitive measure of ectopic recombination, PCR amplificationswere performed on the same samples. The amplification primers wereexpected to produce a 325 bp product from the recombined target and a1.4 kb fragment from the unrecombined allele (FIG. 1). The assay wasexpected to measure the cumulative level of recombination, for any P2Bralleles formed during transient expression of Cre during developmentwould be preserved and perhaps amplified in descendant cells. Low levelsof ectopic recombination product were observed in some tissues of allProCre lines except for one. A Southern blot of PCR amplificationproducts of the P2Br allele utilized tissues from a male heterozygousfor the ProCre nucleic acid construct and the P2Bc allele. DNA from 10different tissues was amplified using primers and conditions thatproduced a 350 bp product from the recombined, P2Br allele. Each lanecontains 10% of the reactions, except for the testis reactions whichwere diluted 500 (T5), 250 (T2) and 100 (T1) fold prior to loading, anda liver reconstruction control (C), which was diluted 1:100 beforeloading. The highest level of ectopic activity was observed in cardiacventricular muscle of mice; in these samples the ectopic signal was morethan 100 fold lower than that observed in testis. Three strains showedmuch lower levels of recombination in brain tissue, and strain 75additionally showed ectopic activity in spleen. Despite the difficultyof quantifying PCR results, these data clearly indicate that ectopicactivity occurred at very low levels in most tissues of most ProCrelines.

Example 5 Isolation of Homologously Recombined ProCre ES Cell ClonesUsing Targeting Vectors with a LoxP-Flanked Selectable Marker

Four male +/ProCre ES cell lines were established from 129/Sv strainProOre transgenic mice. In preliminary experiments, passage 5 cells fromone of these lines (PC3) were used to generate three male chimeras withbetween 50 and 95% coat color chimerism. In matings with C57BL/6females, two of these male chimeras have sired a total of 11 pups, allbearing the Agouti coat color signifying germline transmission of the EScell genome, and 6 of 9 pups genotyped additionally contained the line70 ProCre nucleic acid construct. The frequency of germline transmissionhas not yet been determined, nor has it been determined whethercompetency for germline transmission will persist in homologouslyrecombined ProCre ES cells at later passages.

To determine if homologously recombined ProCre ES cell clones could beisolated using targeting vectors that contained a loxP-flankedselectable marker, two transfections were done using variants of atargeting, vector in which a loxP-flanked neomycin cassette was insertedinto an Nru I site in the hoxb-1 locus promoter (FIG. 2). A Southernblot of BamRi-digested genomic DNAs were harvested from a 96-well platefrom 10 doubly-selected ES cel clones and hybridized with a probe (shownin FIG. 2) which is external to the targeting construct. All samples asshow the 7.5 kb band from the wild-type allele and four clonesadditionally show the 6 kb band predicted to result from homologousrecombination. In these transfections, 12 of 62 (19%) PC3— and 10 of 56(18%) PCs-derived clones that were ganciclovir and G418-resistant(Mansour et al. (1988) Nature 336:348-352) were found to be homologouslyrecombined. In two parallel transfections of CCE cells (Robertson et al.(1986) Nature 323:445-8) with the same vectors, 32 of 93 (34%) and 15 of132 (11%) clones were homologously recombined. The total numbers ofG418-resistant clones recovered from ProCre ES cell transfections werereduced relative to the parallel CCE transfections. This may beattributable to both Cre-mediated excision of the neomycin cassette andto the fact that the transfections were done under electroporationconditions optimized for CCE cells.

Because it was formally possible that the homologously recombined clonescontained inactive loxP sites, five homologously recombined PC3 ES cellclones and the parental PC3 cell line using the primers shown in FIG. 2were either mock transfected or transiently transfected with the pOG231Cre expression vector. For the transient transfection assay, DNA washarvested 48 hours after transfection and used in PCR assays to assesswhether the loxP sites in rice recombinant clones could be recombined byCre. In all cases a clear recombination signal was observed in thepOG231 transfected sample. The recombinant clones and parental celllines show the 204 bp amplification product of the wild-type allele, andthe recombinant clones additionally show, a 1600 bp product (1600)resulting from amplification across the neomycin cassette and anonspecific 1100 bp amplification product (NS). The pOG231-transfectedrecombinant clones show an additional 268 bp product signaling theCre-mediated excision of the neomycin cassette from the recombinantalleles of some cells. Experiments were also done to assess thestability of the loxP-flanked neo cassette in ProCre ES cells. Fiverecombinant clones were grown in the presence of G418 for two weeks, andthen aliquots of each were grown either in the presence or absence ofG418 for a further 10 days. PCR assays were performed to determine ifCre-recombined alleles were present in any of these samples and none wasobserved in the mock transfected controls. These data suggest that thereis not enough Cre activity to significantly influence either the abilityto isolate recombinant clones or the stability of the selectable markersin those clones, establishing that the loxP sites in these clones werefunctional.

To determine if there was any detectable Cre activity in ProCre EScells, aliquots of two lines (PC3 and PC5) were transiently transfectedwith the targeting vector used to create the P2Bc allele. DNA wasrecovered 48 hours after transfection and used for PCR amplifications ofthe P2Br plasmid molecules that would be generated by extrachromosomalCre-mediated recombination. Small amounts of recombination product wereseen in both ProCre ES cell transfections, and none was observed inparallel samples of CCE ES cells. This shows that the ProCre ES celllines express sufficient Cre to recombine some extrachromosomal targetswhen the latter are present at high copy numbers.

Example 6 Plant DNA Constructs

To define sequences in the LAT 52 and LAT59 promoters involved inexpression in pollen, proximal promoters were constructed employing aseries of linker substitution mutants using the particle bombardmentsystem (Klein et al. (1987) Nature 327:70-73; Twell et al. 91989b) PlantPhysiol 91:1270-1274). These experiments were performed by cobombardingthe test plasmids (luciferease [LUC]—recombinase fusions) with referenceplasmids (β-glucuronidase [GUS] fusions). The latter served as a controlfor bombardment variability and allowed comparisons to be made betweenindependent bombardments.

The context of the −100 promoter in LAT52 and the −115 promoter in LAT59was chosen because these promoters appeared to be the minimal regionsthat still conferred high levels (25% relative to the availablefull-length promoter) of pollen-specific expression (Twell et al. (1991)Gen Dev 5:496-507). These minimal promoters were then fused to the Crecoding sequence operatively linked to the luc gene (Ow et al. (1986)Science 234:856-858) coding region, and the resulting plasmids served asa basis for creating the nucleic acid constructs. The LAT52 linkersubstitutions were performed in p52LUC, which contain entire LAT52 5′untranslated region (5′ UTR). A series of six 9- to 10-bp-long linkersubstitutions were made in p52LUC, spanning the region-84 to −29 (52LS1to 52LS6).

Example 7 Tissue Specificity in Plants

The results obtained by transient expression in pollen and in transgenicplants provided information on the effect of the various constructs onexpression in pollen but not on their effect on tissue specificity. Atobacco cell culture, TXD (maintained as described by Howard et al.(1992) Cell 68:109-118), was, therefore, added as an additionalcomponent of the transient assay system. The TXD cell culture wasinitiated from tobacco mesophyll cells and therefore represents somatictissue, as opposed to the gametophytic tissue represented by pollen.Cells in culture were chosen, rather than intact tissue, as the somatictissue source because such cells superficially resemble pollen in thatthey can be spread out as a monolayer on a plate before bombardment.

In this experiment, translation fusions between the Iuc coding regionand either the CaMV 35S promoter drove strong expression in cell culturebut negligible expression in pollen, whereas the LAT52 promoter showedthe opposite pattern of strong activity in pollen and negligibleactivity in cell culture. Thus, the transient assay system mimics theexpression pattern observed for these promoters in transgenic plants(Twell et al. (1991) Genes Dev 5:496-507). This differential expressionprovided us with a tool with which to address tissue specificity.

Example 8 Plant Transformation and Analysis of Transgenic Plants

Constructs cloned into pBin19 were introduced into tomato (Lycopersiconesculentum cv VF36) by Agrobacterium tumnefaciens LBA4404 as previouslydescribed (McCormick (1991b) Transformation of tomato with Agrobacteriumtumefaciens, In Plant Tissue Culture Manual, K. Linsey, Ed B6: 1-9). Atleast 20 independent transformants were obtained for each construct.

For β-glucuronidase (GUS) assays, 5 to 20 μL of pollen, pooled fromseveral flowers of the same plant, was ground directly in Eppendorftubes in 50 to 100 μL of GUS extraction buffer (Jefferson et al. (1987)EMBO 6:3901-3901) using a Teflon-tipped homogenizer driven by a drill.Expression in pollen was measured by fluorometrically assaying GUSactivity in supernatrants of pollen extracts using 2 mM4-methylumbelliferyl d-D-glucuronide (Sigma) as substrate (Jefferson etal. (1987) EMBO 6:3901-3907). GUS activity was corrected for variationin total protein content using a bicinchoninic acid protein assay kit(Pierce, Rockford, Ill.).

Expression in leaves, flowers, stems, roots, and seed was testedhistochemically by staining with 5-bromo-4-chloro-3-indolylβ-D-glucuronide (Molecular Probes, Eugene, Oreg.) as describedpreviously (Jefferson et al. 91987) EMBO 6:3901-3907). Expression inleaves was also analyzed florometrically as given previously.

Example 9 Transient Transformation of tobacco Pollen and Cell Culture

Pollen spread out as a monolayer was bombarded essentially as previouslydescribed (Twell et al. (1991) Genes Dev 5:496-507), except that goldwas substituted for tungsten and only 1 μg of test plasmid and used perplate. TXD cell culture (maintained as described by Howard et al. (1992)Cell 68:109-118) was spread out similarly as a monolayer (1 mL of a50-mL stationary culture per plate) and bombarded as previouslydescribed. Between six and 12 independent bombardments were performedfor each construct. In each experiment, the test plasmid was cobombardedwith a reference plasmid: pB1223 (Clontech, Palo Alto, Calif.) was usedfor assays of all constructs in tobacco cell culture; PLAT59-12 (Twellet al. (1990) Development 109:705-713) for assays of LAT52 and LAT56constructs in tobacco pollen; PLAT56-12 (Twell et al. (1990) Development109:705-713) for assays of LAT59 constructs in tobacco pollen.Processing of the tissue after 15 to 16 hr and analysis of GUS and LUCactivity were as described previously (Twell et al. (1991) Genes Dev5:496-507). Transient expression was reported as “relative LUCactivity,” which represents the ratio between the test (LUC) and thereference (GUS) plasmids.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. A nucleic acid construct comprising a germline-specific promoteroperatively associated with a recombinase coding sequence.
 2. A nucleicacid construct according to claim 1 wherein said germline-specificpromoter is the protamine 1 gene promoter, the protamine 2 genepromoter, the spermatid-specific promoter from the c-kit gene, thesperm-specific promoter from angiotensin-converting enzyme, oocytespecific promoter from the ZP1 gene, oocyte specific promoter from theZP2 gene, or oocyte specific promoter from the ZP3 gene.
 3. A nucleicacid construct according to claim 1 wherein said germline-specificpromoter is the LAT52 gene promoter from tomato, the LAT56 gene promoterfrom tomato, the LAT59 gene promoter from tomato, the pollen-specificpromoter of the Brassica S locus glycoprotein gene, or thepollen-specific promoter of the NTP303 gene.
 4. A nucleic acid constructaccording to claim 1 wherein said recombinase coding sequence encodes arecombinase selected from the group consisting of Cre recombinase, FLPrecombinase, and the R gene product of Zygosaccharomyces.
 5. A nucleicacid construct according to claim 2 wherein said recombinase codingsequence encodes a recombinase selected from the group consisting of Crerecombinase, FLP recombinase, and the R gene product ofZygosaccharomyces.
 6. A nucleic acid construct according to claim 3wherein said recombinase coding sequence encodes a recombinase selectedfrom the group consisting of Cre recombinase, FLP recombinase, and the Rgene product of Zygosaccharomyces.
 7. Plant cells containing a nucleicacid construct comprising a germline-specific promoter operativelyassociated with a recombinase coding sequence, wherein the genomethereof comprises a transcriptionally active selectable marker flankedby two recombination target sites; and wherein the recombinase codingsequence operatively associated with a germline-specific promoter isselective for the recombination target sites flanking said selectablemarker.
 8. A method for the production of recombinant alleles, saidmethod comprising: introducing a nucleic acid fragment flanked by atleast two recombination target sites into embryonic stem cellscontaining a nucleic acid construct comprising a mammaliangermline-specific promoter operatively associated with a recombinasecoding sequence, wherein the nucleic acid construct is in the genome ofthe stem cells and wherein the recombinase is not expressed in the stemcells in cell culture, and passaging the genome derived from saidembryonic stem cells through gametogenesis.
 9. A method according toclaim 8 wherein said nucleic acid fragment comprises an essentialportion of a gene of interest.
 10. A method according to claim 8 whereinsaid nucleic acid fragment is introduced by homologous recombination,random insertion, retroviral insertion or site specific-mediatedrecombination.
 11. A method for the generation of recombinant livestock,said method comprising: combining embryonic stem cells that include anucleic acid construct according to claim 1 with host pluripotential EScells derived from early pre-implantation embryos, and introducing thesecombined cells into a host female and allowing the derived embryos tocome to term.
 12. A method for the production of recombinant alleles ina transgenic non-human animal, said method comprising: introducing anucleic acid fragment flanked by at least two recombinase recombinationtarget sites into mammalian embryonic stem cells containing a nucleicacid construct of claim 1; passaging the genome derived from saidembryonic stem cells through gametogenesis to obtain a transformedgamete; and obtaining progeny from the transformed gamete, therebyproducing a transgenic non-human animal having a recombinant alleletherein.
 13. A method according to claim 12 wherein said nucleic acidfragment is introduced by homologous recombination, random insertion,retroviral insertion, or site specific-mediated recombination.
 14. Amethod for the production of recombinant alleles, said methodcomprising: introducing at least one nucleic acid construct according toclaim 1 into the genome of mammalian embryonic stem cells, wherein saidat least one nucleic acid construct further comprises a nucleic acidfragment flanked by a second pair of recombination target sites and aselectable marker flanked by a first pair of recombination target sites,passaging the genome derived from embryonic stem cells selected forexpression of the marker through gametogenesis to obtain a transformedgamete; and crossing the genome of the transformed gamete with thegenome of a wild type animal, thereby obtaining first generation progenywherein the marker is excised in the germ-line.
 15. A method accordingto claim 14 wherein said first pair of recombination target sites isrecognized by a recombinase which is expressed under the control of agerm-line-specific promoter and said second pair of recombination targetsites is recognized by a recombinase which is expressed under thecontrol of an inducible promoter or a tissue specific promoter.
 16. Amethod according to claim 14 wherein said embryonic stem cells furthercomprise a second nucleic acid construct selected from the groupconsisting of a construct comprising an inducible promoter operativelyassociated with a recombinase coding sequence and a construct comprisinga tissue-specific promoter operatively associated with a recombinasecoding sequence.